Differential expression of metallothioneins in human colorectal cancer specimens and their correlation with p53 protein expression”.

JUAN M ARRIAGA ESTRELLA M LEVY ALICIA I BRAVO SERGIO MORALES BAYO EDUARDO HUERTAS MARÍA M BARRIO MARÍA P ROBERTI MARIANA ARIS JOSE MORDOH MICHELE BIANCHINI

Washington

Congreso; 101st Annual Meeting American Association for Cancer Research AACR; 2010

American Association for Cancer Research

Background and Objective: Metallothioneins (MTs) are a family of low molecular-weight and sulphydryl-rich proteins involved in a wide variety of cellular functions such as the control of p53 activity by intracellular Zn2+ modulation. Several studies have described increased expression of MTs mRNA and protein in various human cancers; conversely, other tumor types, such as colorectal cancer (CRC) and hepatocellular carcinoma, show downregulation of MTs expression during cancer progression. Since the biological significance of MTs  in human colorectal cancer is not fully understood and the expression of its various isoforms is unknown, we analyzed all 9 functional MT 1 and 2 isoforms  (MT1+2) by performing quantitative real-time PCR (qRT-PCR) using total RNA extracted from tissue specimens by laser capture microdissection of epithelial colon cells. In addition, MT1+2 and p53 expression were also evaluated at the protein level by immunohistochemistry using whole tissue sections of both tumoral and normal archival paraffin-embedded colon tissues. Finally, since the expression of the MT1G isoform was found significantly down-regulated in colon cancer cells, we sought to characterize the promoter methylation status of this gene in tissue specimens from CRC and paired normal mucosa by bisulfite treatment and quantitative methylation-specific PCR. Results: the analysis of the epithelial mRNA expression of all 9 functional MT 1 and 2 isoforms by qRT-PCR revealed that most of them were downregulated in tumor epithelium with respect to its paired normal tissue, with the MT1G isoform being significantly more so than the others (median of 100-fold decrease, p < 0.05). Further analysis of methylation levels in the MT1G promoter revealed a significant hypermethylation in tumor samples with respect to paired normal mucosa. We then performed a semiquantitative analysis of MT1+2 and p53 immunostaining: a variable immunoreactivity for MT1+2 was encountered within tumor samples mainly showing reduced MT expression in 90% of tumors. No significant correlations were found when MT staining was analyzed in comparison to histological grading or staging. However, a strong inverse correlation (Pearson r = -0.81) was encountered between MT1+2 loss and p53 overexpression in epithelial tumor cell nuclei. Conclusions: our results confirm that human CRC cells reduce MT1+2 gene and protein expression compared to their paired normal tissue, and demonstrate for the first time that MT1G is the most downregulated isoform. Interestingly, the mechanism leading to MT1G mRNA downregulation involves promoter hypermethylation. Furthermore, MT1+2 and p53 expression seem to be inversely correlated in CRC, a fact that should be further studied to clarify its relevance for carcinogenesis and its possible clinical implications